The invention relates to the field of tumour therapy.
More than 80% of tumours occurring in man are of epithelial origin. The formation of epithelial tumours (carcinomas) is a multi-stage process which is illustrated most clearly in the progression of human colon carcinoma (Powell, et al., 1993) and skin tumours in mice (Wright, et al., 1994). Carcinomas are assumed to start from individual cells or small groups of cells in which mutations have occurred. These cells develop into benign, epithelial hyper- or dysplastic regions. The progression of these hyperplastic regions into a carcinoma in situ, which may then acquire invasive and metastatic properties, requires a number of further mutations in the tumour cell. Characteristically these cells acquire the ability to break down their basal membrane proteolytically, to develop from a stationary polarised cell into a non-polarised cell capable of migrating in the tissue, to survive in the bloodstream and form metastases at remote sites (Liotta, et al., 1991; Liotta and Stetler-Stevenson, 1991).
Although deep changes in gene expression are involved in the many changes in the architecture and behaviour of malignantly transformed cells, none of these newly acquired properties occurs only in invasive tumour cells. Attachment to the basal membrane, proteolysis thereof and migration through the basal membrane and the underlying mesenchyme are important stages in normal processes, e.g. in the implantation of the trophoblast, in movements of configuration during development of the embryo, in the development of the mammary gland and the reorganisation of epithelia during wound healing (Aznavoorian et al., 1993).
For a better understanding of the development and progression of carcinomas it is crucial to understand how the deregulation of these normal processes takes place in cell invasion and metastasisation.
Studies in recent years have contributed to an understanding of the molecular mechanisms involved in the modulation of the epithelial phenotype in normal and pathological situations (Reichmann, et al., 1992; Frisch, 1994). Moreover, exogenous polypeptide factors such as Scatter Factor (SF)/Hepatocyte growth factor (HGF) and New-Regulin/HER-Regulin play important roles in the changes in the migration and differentiation properties of epithelial cells (Birchmeier et al., 1993; Hartmann et al., 1994; Soriano et al., 1995). only recently, Transforming Growth Factor 1 (TGFxcex21) was identified as another potent modulator of the phenotype of breast epithelial cells (Miettinen et al., 1994; Zambruno et al., 1995).
TGFxcex21 belongs to a large super-family of multifunctional polypeptide factors. The TGFxcex2 family itself consists of three genes, TGFxcex21, TGFxcex22 and TGFxcex23, which have extremely high homology with one another. In mammals the TGFxcex2-super-family also includes the various TGFxcex2 genes as well as the embryonic morphogenes, such as e.g. the family of the activins, xe2x80x9cMxc3xcllerian Inhibitory Substancexe2x80x9d, and the bmp family (xe2x80x9cBone Morphogenetic Proteinxe2x80x9d), which play important roles both in regulating embryo development and in the reorganisation of epithelia (Roberts and Sporn, 1992). TGFxcex21 inhibits the growth of many cell types, including epithelial cells, but stimulates the proliferation of various types of mesenchymal cells. In addition, TGFxcex2s induce the synthesis of extracellular matrix proteins, modulate the expression of matrix proteinases and proteinase inhibitors and change the expression of integrins. Moreover, TGFxcex2s are expressed in large amounts in many tumours (Derynck et al., 1985; Keski-Oja et al., 1987). This strong occurrence in neoplastic tissues could indicate that TGFxcex2s are strategic growth/morphogenesis factors which influence the malignant properties associated with the various stages of the metastatic cascade. TGFxcex2s inhibit the growth of normal epithelial and relatively differentiated carcinoma cells, whereas undifferentiated tumour cells which lack many epithelial properties are generally resistant to growth inhibition by TGFxcex2s (Hoosein et al., 1989; Murthy et al., 1989). Furthermore TGFxcex21 may potentiate the invasive and metastatic potential of a breast adenoma cell line (Welch et al., 1990), which indicates the role of TGFxcex21 in the tumour progression. The molecular mechanisms underlying the effect of TGFxcex2s during the tumour cell invasion and metastasisation do, however, require further explanation.
The formation of breast cancer (mammary carcinoma) in humans involves the overexpression of (mutated or, more often, non-mutated) ras-genes and the overexpression of receptor-tyrosinekinases, which activate the Ras-signal transmission pathway (De Bortoli et al., 1985; Kern et al., 1990; LeJeune et al., 1993).
The aim of the present invention was to provide new pharmaceutical compositions for tumour therapy.
The solution to the problem started from the following findings obtained from the tests carried out:
1. The activity of TGFxcex2 on the tumour cell, in cooperation with (i) the expression of oncogenic Ras, with (ii) the overexpression of normal Ras or of receptor tyrosinekinases which activate the Ras signal transmission pathway or with (iii) other oncogenes activated in the tumour cell, lead to a conversion of epithelial cells into fibroblastoid cells with invasive potential.
2. The autocrine production of TGFxcex2 by the converted cells leads to the maintenance of the degenerate, invasive cell status.
3. Interruption of the transmission mediated by the TGFxcex2-receptor signal prevents xe2x80x9cepithelial-fibroblastoid conversionxe2x80x9d (EFC) and the concomitant invasiveness and may change cells which have already undergone an EFC and are growing in a stably invasive manner back into epitheloid cells which are no longer growing invasively (fibroblastoid-epithelial conversion; FEC).
Within the scope of the present invention, the role of TGFxcex21 in the normal development of the mammary glands was investigated with a view to assessing possible side effects of TGFxcex21 inhibitors.
Within the scope of the present invention, it was shown, on the one hand, that Ha-Ras-transformed breast epithelial cells (EpRas-cells) undergo a transition (conversion) from the epithelial to the fibroblastoid (or mesenchymal) state in the formation of tumours in mice. This transition is hereinafter referred to as EF-transition or EF-conversion (xe2x80x9cEpithelial-Fibroblastoid Cell Conversionxe2x80x9d, EFC). Such an EF-conversion has also been demonstrated in vitro. For this, EpRas cells were cultivated in type I collagen gels. In the absence of serum these cells developed into three-dimensional, cystic hollow structures, the walls of which consisted of a single-thickness layer (monolayer) of polarised epithelial cells. TGFxcex21 caused these same Ras-transformed cells to develop into disorganised strands consisting of spindle-shaped cells with fibroblastoid properties. In non-transformed epithelian cells TGFxcex21 was unable to cause such changes. The converted cells were highly invasive both in collagen gels and in chicken heart invasion assays. Surprisingly it was found that, once the fibroblastoid cells had undergone the conversion, they themselves produced large amounts of TGFxcex21. If this self-produced TGFxcex21 was inactivated by a TGFxcex21 neutralising antibody, the cells changed back into a polarised, epithelial phenotype. This cell behaviour indicates that the converted fibroblastoid phenotype is maintained by TGFxcex21, the TGFxcex21 acting through an autocrine loop.
It was also shown, within the scope of the present invention, that the mechanism observed in vitro also applies in vivo: tumour cells which had undergone an EF-conversion themselves produced TGFxcex21. Moreover, TGFxcex21 is capable of triggering and sustaining the invasive phenotype of Ha-Ras-transformed breast epithelial cells in experimentally induced tumours.
Moreover, it was shown within the scope of the present invention that in human tumours of various origins (kidney cell carcinoma, breast cancer) there were indications of the occurrence of xe2x80x9cEpithelial-Fibroblastoid Cell Conversionxe2x80x9d (EFC) (75% of the kidney cell carcinomas investigated and 25-60% of the breast tumours coexpressed the general epithelial marker cytokeratin and the mesenchymal marker vimentin). It was also shown that all these tumours themselves produce TGFxcex21. This is an indication that the results obtained with the model system used in the present invention also apply to human tumours.
Fourthly, it has been shown within the scope of the present invention that total inhibition of the signal transmission induced by the TGFxcex2 receptor can be achieved using a dominant-negative TGFxcex2-receptor chain II (Txcex2RII-dn). Such expression of Txcex2RII-dn led to the elimination of the malignant, invasive phenotype, not only in Ras-transformed mouse-breast epithelial cells, but also in a number of already mesenchymal, invasively growing carcinoma cell lines in humans and mice and to the complete inhibition of the formation of tumours or metastases obtained by these lines in the experimental animals.
The present invention is thus based on the following findings:
Numerous mutations in protooncogenes and tumour suppressor genes participate in carcinogenesis (Vogelstein and Kinzler, 1993). However, little is known about how specific oncogenic mutations are connected with defined changes in the phenotype of the cell and the manner in which these changes then contribute to tumour cell invasion and metastasisation. Within the scope of the present invention it was first demonstrated, using a model system, that the Ras-oncoprotein dramatically changes the cell reaction of breast epithelial cells to TGFxcex21 both in collagen gels and in developing tumours. This modified reactivity of the cells causes TGFxcex21 to induce an EFC. Once converted, these fibroblastoid cells themselves produced high concentrations of TGFxcex21 and thus retained their own mesenchymal and invasive properties.
The theoretical validity of this principle could then be demonstrated in a number of unrelated tumour models in humans and mice. In these tumour cells, other oncogenes very probably take on the function of Ha-Ras. It has been shown that in all these cells both TGFxcex21 and the interruption of any existing autocrine stimulation by TGFxcex21 dramatically influences the tumour cell phenotype: TGFxcex2 also leads to an increase in invasive growth in these cells, whereas switching off the TGFxcex2-receptor or the signal transmission pathways activated by it led to reformation of the EFC, i.e. to a fibroblastoid-epithelial conversion (FEC) and/or to loss of the invasive, tumour-producing cell phenotype.
The experiments carried out within the scope of the present invention originally started from the observation that Ras-transformed mouse breast epithelial cells convert into invasive spindle cells during tumour formation. Similar spindle cell tumours have been described in the brain, skin, colon and breast, both in humans and in animal models (Buchmann et al., 1991; Guldberg, 1923; Sandford et al., 1961; Sonnenberg et al. 1986; Stoler et al. 1993). The origin of these spindle cell carcinomas is still unclear, although some researchers believe that these often highly invasive tumours constitute a separate class of tumour of fibroblastoid origin, whilst other authors assume that these tumours are of epithelial origin.
In the model system used within the scope of the present invention the spindle cell tumours first used clearly originated from the epithelial donor-cells injected into the animal. Spindle cells originating from the tumour survived the selection in G418 and expressed cell- and tissue-specific cytokeratins, confirming their donor cell status and their epithelial origin. Moreover, the tests carried out showed that the injected epithelial cells and the converted fibroblastoid tumour cells came from the same cell clone and re-integration of the retroviral vector into other sites of the genome could be ruled out as a possible cause of the changes. What was almost more important was that the fibroblastoid phenotype of the converted cells was absolutely stable under standard culture conditions and that the cells changed back to polarised epithelial cells efficiently after neutralisation of the TGFxcex21 activity. This rules out genetic or epigenetic changes being responsible for the cell conversion. The most probable explanation for the dramatic change in the phenotype in vivo is that an interaction between the Ras-transformed cells and mesenchymal cells surrounding them leads to the conversion of the epithelial cells into fibroblastoid cells. Within the scope of the present invention it has thus been shown that EFC is a mechanism which is relevant to carcinogenesis in certain tumours.
Within the scope of the present invention it has also been shown that TGFxcex21 induces EFC both in collagen gels and during tumour development. In the cell model used first, this TGFxcex21-induced conversion remarkably requires the cooperation of an activated Ras protein; neither primary breast epithelial cells nor the parental EpH4 cells underwent TGFxcex21-induced EFC. From this it can be concluded that EFC is triggered by a synergy of various signal transmission pathways which are activated on the one hand by TGFxcex21 and on the other hand by Ha-Ras. This assumption is supported by other findings which indicate that activated Ras proteins have similar effects on cells to members of the TGFxcex2 family. This applies e.g. to myogenic differentiation (Payne et al., 1987) and to the formation of the mesoderm (Whitman and Melton, 1992). In the heart muscle, TGFxcex2 highly regulates genes associated with the growth of the embryonic heart regulated by haemodynamic loading. These effects are at least partly mimicked by activated Ras (Parker et al., 1990; Thorburn et al., 1993), leading one to suppose that Ras and TGFxcex2 can act synergistically, at least in some biological systems.
In connection with this it is interesting that the overexpression of normal and mutated Ras was observed in a considerable number of human carcinomas, including breast cancers (De Bortoli et al., 1985; Hand et al., 1984; Slamon et al., 1984). Furthermore, autocrine production of ligands as well as overexpression and/or constitutional activation of receptor-tyrosinekinases which occur at the start of signal transmission pathways including c-Ras (e.g. HER-1, HER-2), are frequent changes in breast cancers (Kern et al., 1990; LeJeune et al., 1993). Since TGFxcex21 is also abundantly present in many human tumours (Derynck et al., 1985; Keski-Oja et al., 1987; Thompson et al., 1991) it may be concluded, on the basis of the results obtained within the scope of the present invention, that Ras- and TGFxcex21-induced signals also act synergistically in human tumours. As demonstrated by the results of the experiments carried out within the scope of the present invention, the TGFxcex2-receptor may also control EFC and invasiveness in tumour cells transformed by oncogenes other than Ras. Another major finding within the scope of the present invention is thus that TGFxcex2 cooperates with various oncoproteins, including Ras, and tyrosinekinases, in regulating the plasticity of the polarised epithelial phenotype.
After serum-free (and TGFxcex2-free) cell culture in reconstituted collagen gels, EpRas-cells exhibited a great capacity for organogenesis and a high degree of epithelial polarisation. The Ep-Ras cells, however, predominantly formed widened tubuli as well as alveolar cavities, in contrast to the narrow branching tubuli formed by the parental EpH4-cells or by primary breast epithelial cells. This shows that the Ras-oncoprotein on its own, in the absence of TGFxcex2, is capable of modifying the morphogenetic behaviour of epithelial cells to some extent. In other systems activated Ras has been described as having more powerful effects on epithelial polarity (Eaton and Simons, 1995). Here, transformation with Ras led to disruption of the polar expression of apical proteins, whilst the expression of basolateral marker proteins remained unaffected (Schoenenberger et al., 1994). Since the experiments described above were carried out in the presence of FCS (which itself contains TGFxcex21), however, it is difficult to compare them with the results obtained within the scope of the present invention in which no such obvious polarity defects could be observed. Since exogenous TGFxcex21 completely destroyed the cell polarity, it is possible that the partial destruction of the polarity in the abovementioned Ras-transformed cell systems can be put down to the TGFxcex21-concentrations present in the serum. Nonetheless the morphogenetic behaviour in EpRas-cells is changed slightly, possibly as the result of increased protease activity.
TGFxcex2 completely destroyed the polarity of epithelial cells and caused the cells to become spindle-shaped and invasive. These changes were dependent on the constant presence of TGFxcex21, since the spindle cells quickly changed back into polarised epithelial cells when TGFxcex21 neutralising antibodies were added. The most important conclusion from these results is that Ras-transformed cells can be switched back and forth by TGFxcex21 between a quasi-normal and a highly tumorigenic phenotype. This intense phenotypical plasticity might be characteristic of invasively growing cells in general and might explain why invasive tumour cells often exhibit the migratory properties of fibroblasts. After extravasation these fibroblastoid, migratory cells should be able to develop back into well differentiated secondary tumours in the new environment provided by remotely located tissue (see below). Increased phenotypical plasticity is thus a characteristic of invasive tumour cells.
Another essential finding reached within the scope of the present invention is that EpRas cells have to undergo EFC in order to produce significant amounts of TGFxcex21 both in vitro and in vivo. It has been shown that tumour cells are able to maintain their fibroblastoid phenotype by means of the autocrine production of TGFxcex21 and that the autocrine TGFxcex21 production and effect on the producing cell (autocrine loop) has to be interrupted in order to make the phenotypical re-conversion of the cells possible. The ability of TGFxcex21 to induce EFC and then efficiently maintain the invasive phenotype may also explain why the initially epithelial Ras-transformed cells changed progressively and uniformly into spindle cells during the tumour growth.
Shortly after their injection into mice, polarised Ras-transformed epithelial cells neither expressed nor released significant quantities of TGFxcex21. As was established by hybridisation in situ and immunohistochemistry, however, the stroma cells surrounding the microtumour expressed the cytokine. These stroma cells could be identified as fibrocyte and endothelial cells, but it must be presumed that other cell types, such as macrophages and lymphocytes, were probably also present; all these cell types are known to produce and release TGFxcex21. The most probable conclusion is that the effects of TGFxcex21 are regulated primarily at the level of their proteolytic activation. The primary regulation of TGFxcex2 is carried out by factors which control the processing of the latent into the biologically active molecule. However, virtually nothing is known about the TGFxcex2 activation in vivo. The protease plasmin can activate two cell types of latent TGFxcex21 in co-culture systems, but only if two different cell types are in direct contact or close together (Antonelli-Orlidge et al., 1989; Sato et al., 1990). This close contact of different cell types should take place in the system used within the scope of the present invention after encapsulation of the tumours by the stroma and to an even greater extent if donor-tumour cells are mixed with stroma cells of the receiver animal during the tumour development (FIG. 2B). Furthermore, thrombospondin (TSP), an extracellular matrix protein, activates latent TGFxcex2. In this case the activation takes place in the soluble phase and requires no proteolytic activity (Schultz-Cherry et al., 1994). In fact, the role of thrombospondin in supporting the development of cancer and the increased thrombospondin concentrations in malignant breast cancers have been briefly reported (Castle et al. 1991; Wong et al., 1992). It has thus been shown within the scope of the present invention that the autocrine production of TGFxcex21, in cooperation with the oncoprotein Ha-Ras, maintains the fibroblastoid phenotype.
The findings and conclusions reached within the scope of the present invention led to the hypothetical model shown in FIG. 9. It is postulated that the TGFxcex21 which is relevant to the tumour formation is produced primarily by infiltrating cells of the tumour stroma, such as fibrocytes, endothelial cells, lymphocytes and macrophages. The interaction of the tumour cells with the different cell types of the tumour stroma should trigger the efficient production and/or activation of TGFxcex21. This should in turn cause the epithelial tumour cells to change into the fibroblastoid and invasive phenotype. These fibroblastoid cells themselves then start to produce TGFxcex21 which acts on them in an autocrine loop and thus both maintains the fibroblastoid phenotype and also makes it easier to recruit other epithelial cells to the EFC. Further mutations or selective mechanisms should cause some of these invasively growing cells to migrate into blood vessels and out again and finally to form secondary tumours at remote sites. This model conforms to findings which show that increased TGFxcex21 expression is also involved in the progression to malignancy in a murine prostate cancer model (Thompson et al., 1992; 1993).
The findings described hitherto were obtained in a combined in vitro/in vivo model system using Ras-transformed mouse-breast epithelial cells. Within the scope of other tests, crucial aspects of this model (EFC, TGFxcex21 production in the tumour) were detected in a large number of primary human carcinomas of the kidneys and breast. Thus, in the majority of all the kidney cell carcinomas investigated as well as in a percentage of the breast tumours investigated dependent on the degree of malignity, the occurrence of an EFC is demonstrated by the coexpression of cytokeratin (general epithelial marker) and vimentin (mesenchymal marker). Moreover, in all the tumours investigated, the production of TGFxcex21 by the tumour cells themselves has been demonstrated both at the protein level by histochemical staining with anti-TGFxcex2-antibodies and also at the mRNA level by in situ hybridisation and RT-PCR.
By means of another series of tests carried out within the scope of the present invention it has been shown that the TGFxcex2-receptor generally assumes a central position in the regulation of EMT and invasive tumour cell growth. Not only in Ha-Ras transformed breast epithelial cells, but in a number of other tumours which originate from other epithelial types and wherein it is not known which oncogenes take over the function of Ha-Ras, the TGFxcex2-receptor has been identified as the crucial regulator of epithelial plasticity as well as of the invasive growth of the tumour cells. Thus, it has been possible to completely inhibit the invasive growth of two human carcinoma cell lines (kidney carcinoma line MZ 1795, nasopharyngeal carcinoma line KB) (presumably caused by secreted TGFxcex21) in collagen gels by means of a neutralising anti-TGFxcex21-antibody.
The proof of the above hypothesis was finally provided by means of a dominant-negative TGFxcex2 receptor (Txcex2RII-dn). This Txcex2RII-dn constitutes a so-called xe2x80x9ckinase-deadxe2x80x9d mutant of the receptor chain II which binds to endogenous receptors of type I, but cannot phosphorylate them. In this way all the Txcex2RII-dn-bound TGFxcex2-receptor chains of type I are inactivated because they cannot activate any signal transmission even after the binding of the ligand (TGFxcex21) since the phosphorylation by receptor chain II required for this is absent. If a dominant-negative TGFxcex2-receptor of this kind is overexpressed in tumour cells, the entire signal transmission proceeding from the TGFxcex2-receptor can be inhibited in these cells. The expression of Txcex2RII is thus suitable for simulating the activity of inhibiting TGFxcex2 or inhibiting the signal transmission pathway triggered by the activation of the TGFxcex2-receptor.
At first Txcex2RII-dn was overexpressed in Ha-Ras-transformed mouse-breast epithelial cells (EpRas). All the clones obtained exhibited greatly delayed tumour growth in nude mice. Moreover, the cells isolated from such tumours had an epithelial phenotype and expressed epithelial markers (E-Cadherin, ZO-1) but no mesenchymal markers (vimentin). This shows that the expression of a Txcex2RII-dn inhibited EFC during tumour formation.
After obtaining these results it was useful to check whether switching off the signal transmission of the TGFxcex2-receptor also works in tumour cells which have already undergone EFC and thus have a stable mesenchymal and invasive phenotype. The colon carcinoma line CT26 in the mouse was selected as an example of such a cell line. This tumour cell line has a very marked tendency to form lung metastases rapidly after subcutaneous injection in mice, so that the animals die from the lung metastases even after the primary tumour has been surgically removed in good time. This cell exhibits mesenchymal morphology, grows into disordered chains and strings of spindle-shaped cells in the collagen gel and expresses no epithelial markers apart from basal cytokeratins. Instead the cells have a high vimentin expression. If the dominant-negative TGFxcex2-receptor (Txcex2RII-dn) is overexpressed in these cells, the cells form smaller or larger compact clumps in the collagen gel and grow on plastic as epitheloid cells which form hemicysts (domes) and express large amounts of E-cadherin and ZO-1. The cells were thus obviously changed back, by the Txcex2RII-dn, into cells with an epithelial phenotype (fibroblastoid-epithelial conversion, FEC).
A corresponding activity of the dominant-negative TGFxcex2-receptor (Txcex2RII-dn) was also observed in vivo. When different, Txcex2RII-dn expressing clones of CT26 cells were injected into mice, the tumour formation was delayed by different amounts depending on the clone. In many clones tumour formation only occurred after 6-8 weeks, as opposed to 1-2 weeks in the case of animals injected with control CT26 cells without Txcex2RII-dn. However, the activity of the Txcex2RII-dn was even more dramatic when the primary tumours were removed from the mice at a certain size and the formation of metastases was expected. In this experiment metastases did not develop in any of the mice injected with Txcex2RII-dn expressing CT26 cells (even after more than 18 weeks) whereas the control animals died of lung metastases within 2-4 weeks after excision of the tumour.
The decisive conclusion from these experiments for the present invention is that inhibiting the signal transmission mediated by the TGFxcex2-receptor can not only prevent the occurrence of an EFC and the resulting acquisition of invasive properties, but also change any existing, invasively growing tumour cells back into a benign state in which they are no longer invasive.
To sum up, the findings obtained within the scope of the present invention indicate that the increased sensitivity and altered reactivity of the cells, compared with the ability of TGFxcex2 to modify the epithelial phenotype, generally represents a characteristic of epithelial tumour cells. This altered reactivity can be brought about by Ras-(onco)proteins, but also by tyrosinekinases which activate Ras, as well as by other as yet unknown oncoproteins. This altered oncogene-induced mode of reaction to normal environmental signals, such as e.g. those induced by TGFxcex21, should lead to altered gene expression in the tumour cell and also incorrect transmission or interpretation of signals between tumour and stroma cells. This abnormal xe2x80x9ccrosstalkxe2x80x9d between tumour cells and their immediate environment would appear to be the driving force for what is commonly known as tumour progression.
Furthermore, the results of the experiments show that activated Ras, overexpressing receptor-tyrosinekinases which activate the Ras signal transmission pathway as well as other, possibly unknown oncogenes co-operate both in the normal development and also in the carcinogenesis with the TGFxcex21-receptor. This would appear to involve processes such as the induction/activation of stromal TGFxcex21 by the interaction of epithelial and mesenchymal cells as well as EF conversion, induced and maintained by TGFxcex21.
The main difference between normal and oncogene-transformed tumour cells should therefore be as follows: TGFxcex21 has a physiological, strictly regulated function during the morphogenesis of normal cells. In the tumour cell the transformation by oncogenes causes degeneration of the function of TGFxcex21, i.e. constitutive, highly abnormal morphogenetic changes are triggered in the cells.
The present invention thus relates to a pharmaceutical composition containing as active compound a substance which inhibits the activity of TGFxcex2 on tumour cells of epithelial origin, for treating epithelial, invasive tumour diseases which are characterised by a reversible transition of the cells from an epithelial, non-invasive state into an invasive state.
In one embodiment of the invention the pharmaceutical composition also contains substances which inhibit the expression of oncogenic Ras and/or the overexpression of normal Ras or the activity of Ras-activating receptor tyrosinekinases in the cells.
In epithelial invasive tumour diseases the tumour cells have an increased phenotypical plasticity, i.e. they are able to undergo transitions from the epithelial, non-invasive state to the fibroblastoid, invasive state (EF conversion) and vice versa (FE conversion).
The substance which inhibits the activity of TGFxcex2 on the cells or the signal transduction mediated by activation of the TGFxcex2-receptor is hereinafter referred to as xe2x80x9cTGFxcex2 inhibitorxe2x80x9d.
TGFxcex2s, like the other members of the TGFxcex2 super-family of multifunctional polypeptide factors such as e.g. activins, Bone Morphogenetic Proteins (bmp""s), etc., exert their effect by binding to specific cell surface receptors. The type I and type II TGFxcex2 receptors form heterodimeric complexes after binding of the ligand, thereby initiating the signal transmission. The type II receptors, which are assigned to the group of the receptor serine/threonine-kinases in terms of their activity, bind the ligands, but require association with the type I receptors which constitute serine-threonine-kinases in order to be able to pass on the signal obtained from the ligand. Whereas the type II receptors are responsible for the ligand specificity, the functionally different type I receptors heterodimerise with several type II receptors. In this ligand-induced heterodimerisation the type II-receptor chains phosphorylate the type I receptors on serine/threonine groups and thereby activate them. This cooperation of the type II-receptor with a particular type I-receptor causes activation of specific signal transmission pathways and as a result leads to a transcriptional response to the signals transmitted to the cell by the ligands.
The activity of a TGFxcex2 inhibitor is based on the fact that it blocks the cell response triggered by the receptor activation, i.e. it prevents the TGFxcex2-receptor system from being activated and hence the cell signal transmission pathway from being actuated.
Since it is the type I receptors which are responsible for the specific transcriptional response which eventually produces the fibroblastoid phenotype after the binding of the ligands to the type II receptor, and to the type I receptor, the type I receptor represents one of the target molecules for the TGFxcex2 inhibitor. Because of the need for phosphorylation of the type I receptor by the type II receptor (and on the basis of the results obtained with dominant negative type II receptors whose serinekinase activity has been destroyed by mutation, the type II receptor is also a possible target molecule for inhibitors.
Other mechanisms for the activity of a TGFxcex2 inhibitor are thus based on preventing the interaction between the ligand TGFxcex2 and the type II receptor, preventing the signal transmitted from the type II receptor to the type I receptor which brings about the activation of the type I receptor. Finally, blocking the binding of TGFxcex2 to the type I receptor, inhibiting the activity of the type I receptor or inhibiting an effector molecule of the signal transmission pathway activated by the type I receptor are all possible methods of attacking inhibitors.
Examples of inhibitors are antibodies which neutralise the TGFxcex2, particularly monoclonal antibodies, TGFxcex2 antisense-RNA molecules (Fakhrai et al., 1996) or dominant-negative TGFxcex2 receptors of type I or II.
The invention relates, according to a further aspect, to screening processes for identifying pharmacologically active substances for treating epithelial, invasive tumour diseases which are characterised by a reversible transition of the cells from an epithelial, non-invasive state into an invasive state.
One method of finding suitable, particularly low-molecular, inhibitors, comprises determining, in a first step, which of the type I receptors is responsible for the transition from the epithelial to the fibroblastoid state of the cells. To do this, the EpRas-cell line used within the scope of the present invention (or one of the other cell lines used which are capable of bringing about the EF conversion or have already undergone one), is interrogated to see which TGFxcex2-type I/II receptor it expresses. This interrogation may be carried out by RT-PCR (xe2x80x9cReverse Transcriptase Polymerase Chain Reactionxe2x80x9d) using oligonucleotides, derived from known TGFxcex2-type I or type II receptors, as PCR primers in order to amplify the relevant receptor-DNA from EpRas-DNA and thus identify the TGFxcex2-type I or type II receptor expressed in these cells. The experiments described in the Examples with a dominant-negative mutant of the human type II receptor Txcex2R-II (only this chain occurs in all the known receptors for TGFxcex21,2,3; Wrana et al., 1992, Wrana et al., 1994) confirm that this TGFxcex2-type II receptor subtype (as such) is necessary, directly or indirectly, for the signal transmission which leads to the EF conversion. This TGFxcex2-type II receptor subtype thus constitutes one of the target molecules for the TGFxcex2 inhibitor. This satisfies an esential precondition for establishing a cellular or biochemical screening assay which can be used to screen specifically for substances which inhibit this target molecule.
Next, an investigation is carried out in a cell which is undergoing the EF conversion or in which the EF conversion can be reversed by inhibiting the TGFxcex2 receptor signal transmission to determine which of the processes taking place in the cell by EF conversion or reversal thereof is most suitable for establishing a screening assay. Appropriately the EpRas cells used in the Examples or the CT26 cells which are also well characterised within the scope of the present invention may be used.
The effects to be expected can be divided into two groups: the first group includes the effects of TGFxcex2 on normal mesenchymal and epithelial cells, e.g. in wound healing, described in the literature. The second group of changes are those which occur in particular with the activity of TGFxcex2 on transformed cells (such as e.g. in the experiments described within the scope of the present invention). Whereas the TGFxcex2 effects of the first group can be adduced for a primary HTS screen (xe2x80x9cHigh Throughput Screenxe2x80x9d), any inhibitor candidates found must be tested without fail for their inhibiting activity on the TGFxcex2 effects of the second group.
The TGFxcex2 effects of the first type include i) the induction of extracellular matrix proteins, such as fibronectin, laminin, elastin; ii) the induction of the protease inhibitor PAI (Plasminogen Activator Inhibitor) and hence the inhibition of cell protease activity, and iii) an inhibition of cell growth and induction of programmed cell death (apoptosis) in certain cell types. These include in particular normal epithelial cells as well as only slightly degenerate, essentially still epitheloid tumour cell lines. The induction of PAI-1 expression as well as the TGFxcex2-induced apoptosis are possible procedures which may be used to design a cell assay system for screening substances which may act as TGFxcex2 receptor inhibitors. The effect chosen is used directly as a system for demonstrating the inhibiting activity of the substance.
In order to find out whether the EF conversion in the EpRas-cell line used within the scope of the present invention triggered by the activation of the TGFxcex2 receptor system is transmitted via the same type I/type II receptors as the induction of PAI (or another molecule regulated by TGFxcex2) in untransformed cells, e.g. in the normal starting cell line EpH4 (also used within the scope of this invention), it is possible to check e.g. whether the induction of PAI (or another molecule) or the growth inhibition which is very marked in this cell line is blocked by a dominant-negative mutant of the same type I or II receptor which also blocks the EF conversion. In the case of the CT26 cells which overexpress the dominant-negative type II receptor (Txcex2RII-dn), it has been shown that, in the Txcex2RII-dn expressing CT26 clones which reverted to epithelial cells, activation of a PAI-1 promoter-controlled reporter gene was completely inhibited by TGFxcex2-1. The extent of the PAI-I inhibition of the inhibition of reporter gene expression by PAI-I correlated directly with the ability of the different clones to form tumours in the animal. Moreover, a special CT26 clone, which had recovered complete TGFxcex2-1-inducibility of the PAI-1 promoter-reporter gene construct after lengthy passage in vitro (presumably by repressing the Txcex2RII-dn expression) also regained the ability to form metastasising tumours in the mouse.
The confirmation of the correlation between EFC, tumour formation and TGFxcex2 receptor type II function using the Txcex2RII-dn experiments provides the prerequisite for a screening assay based on a PAI-1 reporter gene test cell. This test cell, which is a human or animal cell, is stably transformed with a plasmid, in which a reporter gene, e.g. the luciferase gene, is under the control of the regulatory sequence of the PAI gene (or a gene which codes for another molecule regulated by TGFxcex2, e.g. for an extracellular matrix protein). The test cell is also transformed with the human type I or type II receptor, which was shown, after further tests, to be most efficient both at triggering the EF conversion and also at inducing PAI or another molecule regulated by TGFxcex2. The human TGFxcex2 type II receptor used for the construction of the Txcex2RII-dn is one of the possible target molecules for a TGFxcex2 inhibitor. The control cell used is expediently a parallel-cell clone in which the PAI-1-promoter controlled reporter gene is activated by another receptor not related to the TGFxcex2 receptor (e.g. members of the FGF (fibroblast growth factor) receptor-tyrosinekinase family).
If a substance which wholly or partially inhibits the TGFxcex2-induced reporter gene expression is found in a screening assay of this kind, it can be concluded that either the selected ligand-activated type I/type II receptor or the signal transmission mediated by this receptor is blocked by this substance. The same substance should not have any influence on the slight basal reporter gene expression in the control cell in which the reporter gene has been activated not by TGFxcex2, but by FGF. The test systems in which the reporter gene activation is measured can be used in robotised High Throughput Screen (HTS) processes.
A second possible way of measuring the blocking of the TGFxcex2 receptor function by test substances can easily be measured by the removal of the growth inhibition and apoptosis brought about by TGFxcex2. Since TGFxcex2 efficiently induces apoptosis in normal EpH4 cells under certain conditions, effective inhibitors of the TGFxcex2 receptor should act as survival or growth stimulating factors. EpH4 cells in which another apoptosis-inducing receptor has been expressed may be used as control cells. The Fas receptor, which efficiently induces apoptosis in virtually all cell types after the binding of a special Fas ligand, is particularly suitable. The removal of an apoptotic effect by effective TGFxcex2 receptor inhibitors has the advantage that it can easily be measured in commercially obtainable test systems (e.g. in the MTS assay which detects the number of live, metabolically active cells), and that toxic substances (which cause rather than prevent cell death) can easily be identified as such. Thus, this test system is also suitable for HTS primary screens.
Another possible cell assay system with which substances can be tested for their inhibiting activity on the EF conversion triggered by activation of the TGFxcex2 receptor system, is based on the expression of proteins which are characteristic of the fibroblastoid cell type after EF conversion and are thus an indicator of the occurrence of EF conversion. One example of this is vimentin (Reichmann et al, 1992): it has been shown within the scope of the present invention that expression thereof goes hand in hand with the EF conversion triggered by cooperation of Ras and TGFxcex2. Other examples of other markers of the fibroblastoid phenotype are the loss of the expression of E-Cadherin mRNA as well as the de-novo expression of fibronectin and diverse proteases (UPA, TPA, Reichmann et al, 1992). A suitable test cell transformed by Ras or another oncogene is transformed with a plasmid in which a reporter gene is under the control of the vimentin gene promoter or of promoters of one of the other fibroblastoid marker genes mentioned. The modulation of the reporter gene expression by a test substance should then correlate with the modulation of the EC conversion brought about by the same inhibitors.
Another possible way of finding substances which inhibit the activation of the TGFxcex2 receptor system uses the expression of TGFxcex2 itself as a detection system. This assay principle is based on the finding reached within the scope of the present invention that the activation of the TGFxcex2 receptor system in oncogene expressing cells by the ligand TGFxcex2 causes the autocrine production of TGFxcex2 which acts on the cells in an autocrine loop. In an assay of this kind, which can detect both the activation of the TGFxcex2 receptor system and also the induction of the autocrine TGFxcex2 loop brought about by the expression of Ras (the activity of substances which inhibit TGFxcex2 expression, in a test of this kind, do this on the basis of their effect on the activation of the TGFxcex2 receptor system and their effect on Ras), the cells contain a reporter gene construct which is under the control of the TGFxcex2 gene promoter (Kim et al, 1989).
Biochemical assays in which TGFxcex2 inhibitors are identified, the activity of which is based on the fact that they inhibit the TGFxcex2 signal transmission pathway, may be carried out as follows, for example: in an assay format the autophosphorylation of the TGFxcex2 receptor type II or the cytoplasmic domain thereof which contains the kinase domain is measured in vitro on serine or threonine groups, in the presence and in the absence of test substances (potential TGFxcex2 inhibitors), a kinase assay of this kind being carried out using methods known from the literature, e.g. as described by Lin et al., 1992, or Braunwalder et al., 1996, and using a receptor (or a domain thereof) prepared by recombinant methods, e.g. in E. coli. In an alternative assay format, the ability of the TGFxcex2 receptor type II to phosphorylate the TGFxcex2 receptor type I or its so-called GS domain (Wrana et al., 1994), is measured, again according to the known principle of kinase assays, in the presence and absence of potential inhibitor. The modification of an assay of this kind for a High Throughput Format can be carried out using commercially available technologies such as filter plates, FLASH plates (Amersham) or SPA (Scintillation Proximity assay)-Beads (Amersham).
In one of the test systems described, inhibitors of the TGFxcex2 receptor found in the primary screen are expediently tested for their specificity in secondary screens. This can be done particularly by direct inhibition of the TGFxcex2-dependent EF conversion of EpRas cells in collagen gels. Another possibility is the incubation of converted EpRas cells (e.g. from mouse tumours) plated out at low density on plastic dishes with the inhibitor of the TGFxcex2 receptor found. Effective substances should trigger the conversion of fibroblastoid into epithelial cells even in the presence of TGFxcex2. The same substances should cause re-epithelialisation (FE conversion) in CT26 cells. Finally, particularly suitable active substances in mice which were injected with CT26 cells can be tested to see whether they slow down the growth of the primary tumour or metastasisation after excision of the primary tumour.
The substance which inhibits the expression or the function of oncogenic Ras and/or the overexpression of normal Ras (or the consequences of this overexpression) and/or the activation of normal Ras by receptor tyrosinekinases in the cells is hereinafter referred to as xe2x80x9cRas inhibitorxe2x80x9d.
Ras inhibitors for the purposes of the present invention either inhibit Ras directly, by inhibiting the activation/function of Ras itself or by inhibiting the activation/function of a Ras-effector molecule which acts below Ras in the Ras signal transmission pathway. Examples are inhibitors of Raf, such as Raf antisense-oligonucleotides (Monia et al., 1996). For cases where the activation of Ras cannot be put down to a change in Ras itself, but it due to the constitutive activation of receptor-tyrosinekinases acting above Ras, inhibition of Ras-activation can also be brought about by inhibiting these receptors. Examples of receptors of this kind are the receptor-tyrosinekinases EGF receptor (xe2x80x9cEpidermal Growth Factor Receptorxe2x80x9d) and homologous receptors such as HER-2, HER-3 or HER-4. Examples of chemical compounds which inhibit the EGF receptor can be found in WO 96/07657. Known Ras inhibitors are monoclonal antibodies (Furth et al., 1982), dominant-negative mutants (Stacey et al., 1991; Quilliam et al., 1994) and antisense-RNA. Examples of low-molecular Ras inhibitors are inhibitors of Ras-Farnesyl transferases (Kohl et al., 1993; Kohl et al., 1994; Kohl et al., 1995).
In order to screen for other low-molecular Ras inhibitors, genes coding for mutations of the Ras proteins H-Ras, K-Ras or N-Ras, which lead to constitutive activation of Ras, are introduced into mammalian cells, e.g. by means of retroviral vectors, and the selective cytotoxic activity of test substances on the ras-transformed cells is determined. A suitable method of identifying ras inhibitors is described e.g. in der EP-A 604 181.
Examples of Ras-transformed cell lines which may be used as test cells for the identification of Ras inhibitors, have also been described by Andrejauskas and Moroni, 1989, as well as by Jenkins et al., 1993.
Ras inhibitors can also be identified with an assay based on the EpRas-cell line used within the scope of the present invention. For this, the cells contain a reporter gene construct in which the reporter gene is under the control of the regulatory sequence of the TGFxcex2 gene. First of all TGFxcex2 is applied to the cells in order to bring about the EF conversion. Then the cells are treated with the test substances. Test substances which are capable of inhibiting the activity of the reporter gene can be assumed to be Ras inhibitors. This can then be confirmed in secondary screens in which the substances are investigated to see whether they can inhibit the TGFxcex2-induced EF conversion of EpRas cells in collagen gels or reverse the EFC which has already taken place.
The pharmaceutical compositions according to the invention can be used, firstly, to prevent the cells from changing into the fibroblastoid state and becoming invasive, thus preventing or reducing their tumorigenicity. Secondly, the pharmaceutical compositions according to the invention can also be used to bring about the conversion of existing fibroblastoid and invasively growing tumour cells into non-malignant or less malignant epithelial cells.
The pharmaceutical composition according to the invention may be used on the one hand to prevent the transformation of the cells from the epithelial, non-invasive state into a fibroblastoid, invasive state. One example of this is its administration after surgical removal of a primary tumour to prevent any tumour cells present from becoming invasive and producing further tumours by metastasisation. Moreover the pharmaceutical composition according to the invention can also slow down tumour growth by the same mechanism, as has been shown with the aid of the Txcex2RII-dn expressing CT26 cells.
The pharmaceutical composition according to the invention may, on the other hand, be used to reverse an EF conversion of the cells which has already taken place. Once the conversion has taken place, TGFxcex2 maintains the fibroblastoid state by means of an autocrine loop. The administration of a TGFxcex2 inhibitor on its own in this case switches off the autocrine loop and thus reverses the fibroblastoid, invasive state of the cell into the normal, epithelial state. However, this reversal is temporary, and there is no fundamental change to the transformed state of the cell brought about by Ras or other oncogenes. This means that when the TGFxcex2 inhibitor is removed the EF conversion could start up again. If, on the other hand, an oncogene inhibitor, e.g. a Ras inhibitor or an HER-1/2 inhibitor, is administered, possibly in addition to the TGFxcex2 inhibitor, the transformed state of the cell is cancelled, the cell behaves like a normal epithelial cell and reacts correspondingly normally to TGFxcex2, i.e. the effect of TGFxcex2 on the cell cannot bring about EF conversion and even leads to growth inhibition of the tumour cell.
The conjecture that TGFxcex2 (receptor) inhibitors could cause slowing down or even inhibition of tumour growth is supported by the following state of affairs: most tumours constantly produce TGFxcex2 (see below) which is released into the environment and has an immunosuppressant effect there, i.e. inhibits the function of cytotoxic T-lymphocytes and other cells of the immune system. If the TGFxcex2 receptor inhibitor causes the transformation of invasive tumour cells into non-invasive, more epithelial cells, these should switch off the secretion of TGFxcex2 and thus be more easily attacked and lysed by cytotoxic T cells.
In order to achieve optimal activity, the pharmaceutical composition according to the invention preferably contains a combination of TGFxcex2 inhibitor and Ras inhibitor.
In the transition of epithelial cells into the fibroblastoid state, fibroblastoid marker proteins, e.g. vimentin, are expressed more intensely. The increase in the expression of these markers (see below) is thus one of the diagnostic parameters for tumour diseases which can be treated using the pharmaceutical composition according to the invention.
These tumour diseases include adenocarcinomas of the breast (Heatley et al., 1993), kidney cell carcinomas (Beham et al., 1992), carcinosarcomas of the breast (Wargotz and Norris, 1989), carcinosarcomas of the oesophagus (Guarino et al., 1993) or of the female genital tract (de Brito et al., 1993), epitheloid sarcomas as well as spindle cell carcinomas of various locations, e.g. lung carcinomas with spindle cell components (Matsui et al., 1992) or spindle cell carcinomas of the gall bladder (Nishihara et al., 1993).
The pharmaceutical composition according to the invention is preferably used to treat breast tumours and kidney cell carcinomas.
The pharmaceutical compositions according to the invention are administered to humans in doses of 0.01 to 100 mg/kg body weight, preferably 0.1 to 15 mg. Apart from the active compounds the pharmaceutical composition contains the usual inert carriers and excipients. The skilled person will find methods of formulating pharmaceutical preparations in the relevant textbooks, such as Remington""s Pharmaceutical Sciences, 1980.